Methods and systems for biological reagent placement

ABSTRACT

An apparatus for placing at least one biological reagent at a plurality of locations on a substrate includes a stamp member onto which the at least one biological reagent is applied. The stamp member defines a plurality of transfer elements patterned to correspond to the plurality of locations. The stamp member contacts the substrate to transfer the at least one biological reagent from the plurality of transfer elements to the plurality of locations. The transfer elements can be defined by reservoirs or projected portions of the stamp member.

This is a continuation of application Ser. No. 08/648,635 filed on May13, 1996 now U.S. Pat. No. 5,731,157.

FIELD OF THE INVENTION

The present invention relates to methods and systems for placingbiological reagents on a substrate.

BACKGROUND OF THE INVENTION

Recently, an increased effort has been directed toward the developmentof chips for molecular detection. In general, a molecular detection chipincludes a substrate on which an array of binding sites is arranged.Each binding site (or hybridization site) has a respective molecularreceptor which binds or hybridizes with a molecule having apredetermined structure. A sample solution is applied to the moleculardetection chip, and molecules in the sample bind or hybridize at one ormore of the binding sites. The particular binding sites at whichhybridization occurs are detected, and one or more molecular structureswithin the sample are subsequently deduced.

Of great interest are molecular detection chips for gene sequencing.These chips, often referred to as DNA chips, utilize an array ofselective binding sites each having respective single-stranded DNAprobes. A sample of single-stranded DNA fragments, referred to as targetDNA, is applied to the DNA chip. The DNA fragments attach to one or moreof the DNA probes by a hybridization process. By detecting which DNAprobes have a DNA fragment hybridized thereto, a sequence of nucleotidebases within the DNA fragment can be determined.

A number of approaches have been devised for putting an array ofmolecular receptors on a substrate. Affymax has proposed a lithographictechnique of synthesizing peptides or nucleic acids on a glass surface.To synthesize an array of n-mer oligonucleotide probes, 4n lithographicwrite steps are required. This results from the four differentconstituent nucleotides (adenine, cytosine, guanine, and thymine) whichcan be located at each of the n nucleotide locations in an n-mer probe.A shortcoming of the lithographic technique is that a new set oflithographic masks must be produced if a new configuration of probes isdesired in the array. Further, the use of 4n mask levels results in anundesirably low yield.

Currently, a molecular sample is applied to probes on a moleculardetection chip by immersing the chip into a sample solution. Thisapproach requires a relatively large quantity of samples whose quantityis often limited. Further, molecules will often bind erroneously tosites on the molecular detection chip to produce false positivereadings.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is pointed out with particularity in the appended claims.However, other features of the invention will become more apparent andthe invention will be best understood by referring to the followingdetailed description in conjunction with the accompanying drawings inwhich:

FIG. 1 is a block diagram of an embodiment of an apparatus for placingat least one biological reagent at a plurality of locations on asubstrate;

FIG. 2 is an illustration of an embodiment of a stamp member inaccordance with the present invention;

FIG. 3 illustrates at least one biological reagent being transferred toa substrate from the stamp member of FIG. 2;

FIG. 4 is an illustration of a rectangular-shaped reservoir for use as atransfer element;

FIG. 5 is an illustration of a spherical-shaped reservoir for use as atransfer element;

FIG. 6 is an illustration of a means for controlling a temperature in areservoir;

FIG. 7 is an illustration of another technique to introduce the at leastone reagent into the reservoirs;

FIG. 8 is an illustration of an embodiment of a stamp member whichprovides another technique for applying the at least one reagent intothe reservoirs;

FIG. 9 is an illustration of a stamp member having a plurality ofprojected portions which define a plurality of transfer elements;

FIG. 10 illustrates an embodiment of the stamp member having a supportmember which supports an absorbent layer;

FIG. 11 illustrates an alternative approach to increasing an absorbencyof the stamp member by increasing a surface area of a stamping surface;

FIG. 12 illustrates an embodiment the stamp member which utilizes bothprojected portions and reservoirs;

FIG. 13 illustrates an embodiment of a stamp member having selectivelyprojectable transfer elements;

FIG. 14 illustrates the stamp member having an unprojected piston and aprojected piston;

FIG. 15 illustrates an embodiment of a selectively projectable stampmember having means for temperature control;

FIG. 16 shows an example of a stamp member having a cylindrical shape;

FIG. 17 illustrates a roller used to apply a reagent to a stamp member;

FIG. 18 illustrates at least one dispensing head used to apply a reagentto a stamp member; and

FIG. 19 is a flow chart summarizing steps performed in an embodiment ofmethod of placing at least one biological reagent at a plurality oflocations on a substrate.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

FIG. 1 is a block diagram of an embodiment of an apparatus for placingat least one biological reagent 100 at a plurality of locations 102 on asubstrate 104. The apparatus includes a stamp member 106 onto which theat least one biological reagent 100 is applied. The stamp member 106defines a plurality of transfer elements 108 patterned to correspond tothe plurality of locations 102. The at least one biological reagent 100is transferred from the plurality of transfer elements 108 to theplurality of locations 102 by contacting the stamp member 106 with thesubstrate 104.

The at least one biological reagent 100 can include a common biologicalreagent which is applied to each of the plurality of transfer elements108. In this case, the common biological reagent is transferred to allof the plurality of locations 102 on the substrate 104, and hence, iscommon to all of the plurality of locations 102. Alternatively, each ofthe plurality of transfer elements 108 can receive a respective one ofthe at least one biological reagent 100. Here, different biologicalreagents can be transferred to different locations on the substrate 104.

It is noted that the at least one biological reagent 100, in thisembodiment and other embodiments described herein, can generallycomprise any reagent or chemical. Of particular interest, however, arebiological reagents including, but not limited to, single nucleotides,nucleotide chains such as DNA fragments and RNA fragments, and othernucleic acids.

Embodiments of the present invention can be advantageously utilized fortransferring biological reagents to a substrate which forms a moleculardetection apparatus, such as a gene sequencing chip or a diseasediagnosis chip. The biological reagents can be transferred to formmolecular detection probes at binding sites of the molecular detectionapparatus. Alternatively, the biological reagents can include targetmolecules which hybridize with selective ones of the binding sitesalready having molecular detection probes.

It is also noted that the substrate 104 can be formed of a variety ofmaterials which include, but are not limited to, metals, semiconductors,paper, glass, and plastic, for the various embodiments of the presentinvention described herein. The substrate 104 can be either flexible orrigid, and can have any shape, including but not limited to a planarshape, a roll shape, and a cylindrical shape.

FIG. 2 is an illustration of an embodiment of a stamp member 120 inaccordance with the present invention. The stamp member 120 defines aplurality of transfer elements 122. The plurality of transfer elements122 can be patterned as an array, such as the two-dimensional arrayillustrated in FIG. 2, to correspond to an array of locations on asubstrate. It is noted, however, that alternative patterns of theplurality of transfer elements 122 can be utilized. The stamp member 120has a face 124 at which the plurality of transfer elements 122 isdefined. In this embodiment, the face 124 is substantially planar.

FIG. 3 illustrates at least one biological reagent being transferred toa substrate 130 from the stamp member 120 of FIG. 2. The face 124 of thestamp member 120 is directed toward a surface 132 of the substrate 130until contact is made. At contact, either the face 124 contacts thesubstrate 130, or at least one of the plurality of transfer elements 122contacts the substrate 130. Thereafter, as indicated by referencenumeral 134, the stamp member 120 is removed from the substrate 130.Here, either the at least one biological reagent or a reaction productthereof remains at a plurality of locations 138 of the substrate 130.

In the various embodiments of the present invention, the plurality oftransfer elements can have any of a variety of forms. In one form, theplurality of transfer elements includes a plurality of reservoirsdefined on a surface of the stamp member. The plurality of reservoirsare arranged to correspond to the plurality of locations on thesubstrate. Each reservoir has the form of a small inclusion in the stampmember for holding small quantities of reagents for chemical reactions.For example, the reservoirs can be utilized to contain on the order of apicoliter of a reagent.

Using conventional aligning techniques for mask/wafer positioning, atleast one reagent can be accurately placed at predetermined locations ofa chemically-sensitive substrate by contacting the substrate with astamp member having a corresponding array of reservoirs. While the stampmember is in contact with the substrate, any chemical reactions betweenthe at least one reagent and the chemically-sensitive substrate arecontained within the reservoirs. Preferably, the reservoirs have apredetermined shape and a predetermined volume so as not tosignificantly affect the chemical reaction of interest.

The stamp member is held in contact with the substrate until thechemical reactions have completed. Thereafter, the stamp member isremoved from the substrate leaving regions of reaction products on thesubstrate.

FIG. 4 is an illustration of a rectangular-shaped reservoir 140 for useas a transfer element. The rectangular-shaped reservoir 140 is definedwithin a portion of a stamp member 142. In one embodiment, therectangular-shaped reservoir 140 has a dimension 144 greater than 10 μmso as not to affect a predetermined chemical reaction performed therein.

FIG. 5 is an illustration of a spherical-shaped reservoir 150 for use asa transfer element. The spherical-shaped reservoir 150 is defined withina portion of a stamp member 152. Similar to the rectangular-shapedreservoir 140, the spherical-shaped reservoir 150 can have a diameter154 greater than 10 μm so as not to affect a predetermined chemicalreaction performed therein.

FIG. 6 is an illustration of a means for controlling a temperature in areservoir 160. A heating element 162 is embedded in a stamp member 164proximate to the reservoir 160. The heating element 162 can be aresistive heating element, for example, which heats the reservoir 160 independence upon a current applied therethrough.

A temperature probe 166 is embedded in the stamp member 164 proximate tothe reservoir 160. The temperature probe 166 measures the temperature ofthe reservoir 160 or of a reagent in the reservoir 160. A signalrepresentative of the temperature is communicated from the temperatureprobe 166 to a controller 168. The controller 168 processes the signalto form a control signal which is applied to the heating element 162.The control signal is formed in order to control the temperature to adesired level.

By embedding a heating element and a temperature probe in each of aplurality of reservoirs, the temperature at each reservoir can belocally controlled. This is useful for controlling hybridization ofgenetic material onto specific substrate locations for diagnosticpurposes.

Temperature control at a reservoir is also beneficial for providing ameans for amplifying a biological sample contained therein. Here, thetemperature can be controlled to provide the heating steps in a PCR(polymerase chain reaction) amplification technique.

For the various embodiments of the present invention described herein,the at least one biological reagent can be introduced into thereservoirs by a number of different techniques. One technique is toprovide a substantially uniform wetting of the stamp member. Here, thestamp member can be immersed in a fluid containing the at least onereagent to fill each of the reservoirs. Alternatively, the face of thestamp member can be rolled on or contacted with a saturated absorbentmaterial containing the at least one reagent. In both of these cases,the same at least one reagent is typically applied to all of thereservoirs.

FIG. 7 is an illustration of another technique to introduce the at leastone reagent into the reservoirs. Here, the at least one reagent isdirectly placed into a plurality of reservoirs of a stamp member 170using a corresponding plurality of capillaries 172 (or a correspondingplurality of micropipets). As illustrated, the at least one reagent canbe applied to the reservoirs from the bottom of the stamp member 170.

FIG. 8 is an illustration of an embodiment of a stamp member 180 whichprovides another technique for applying the at least one reagent intothe reservoirs. A plurality of capillaries 182 or other like tubemembers is embedded in the stamp member 180. A capillary 184 of theplurality of capillaries 182 is illustrated in more detail. Thecapillary 184 is embedded in the stamp member 180 proximate to areservoir 186. The capillary 184 provides fluidic communication to thereservoir 186. Using this embodiment, a respective biological reagentcan be applied to each of the plurality of reservoirs by a respectiveone of the plurality of capillaries 182.

The direct placement approaches illustrated in FIGS. 7 and 8 areadvantageous over the uniform wetting approaches with respect to bothreagent quantity and placement. Specifically, a reduced amount ofreagent is needed to fill the reservoirs using direct placement.Further, different reagents can be applied to different reservoirs forplacement at different locations on the substrate.

As described earlier, the plurality of transfer elements in accordancewith the present invention can have any of a variety of forms. A secondform for the transfer elements is illustrated in FIG. 9.

FIG. 9 is an illustration of a stamp member 190 having a plurality ofprojected portions 192 which define a plurality of transfer elements.The plurality of projected portions 192 are patterned to correspond to aplurality of locations on a substrate at which one or more reagents areto be placed.

The plurality of projected portions 192 absorb at least one biologicalreagent 194 which is applied to a face 196 of the stamp member 190. Asillustrated, the at least one biological reagent 194 can also beabsorbed by non-projected portions 198 of the stamp member 190. This canoccur if the at least one biological reagent 194 is applied using auniform wetting technique as described earlier.

The stamp member 190 is contacted with a substrate 200 to transfer theat least one biological reagent 194 to a plurality of locations 202. Atcontact, a stamping surface 204 of each of the plurality of projectedportions 192 is in contact with the substrate 200 at a respective one ofthe plurality of locations 202. However, the non-projected portions 198do not contact the substrate 200. Upon removing the stamp member 190from the substrate 200, the at least one biological reagent 194 remainsat the plurality of locations 202 on the substrate 200.

The stamp member 190 can take the form of a patterned rigid orsemi-rigid plate fabricated from materials which include, but are notlimited to, glass, elastomer, metal, sol-gels, plastic, and polymers.One or more of these materials can be combined in the stamp member 190to obtain a flexibility, rigidity, opacity, thermal conductivity, and/orchemical absorbency which is desired.

FIG. 10 illustrates an embodiment of the stamp member 190 having asupport member 210 which supports an absorbent layer 212. The supportmember 210 is formed of a rigid or semi-rigid material, such as thoseaforementioned. The absorbent layer 212 is comprised of an absorbentmaterial such as a sol-gel. The absorbent layer 212 defines a pluralityof projected portions 214 patterned to correspond to a plurality oflocations on a substrate.

FIG. 11 illustrates an alternative approach to increasing an absorbencyof the stamp member 190 by increasing a surface area of a stampingsurface. The stamp member 190 defines a plurality of projected portions220. Each of the plurality of projected portions 220 has a stampingsurface 222 which defines at least one cavity 224. The at least onecavity 224 increases the absorbency of each of the plurality ofprojected portions 220 by increasing a surface area of the stampingsurface 222. The at least one cavity 224 can be defined by a series ofridges at each of the plurality of projected portions 220.

FIG. 12 illustrates an embodiment the stamp member 190 which utilizesboth projected portions and reservoirs. Here, each of a plurality ofprojected portions 230 has a stamping surface 232 which defines areservoir 234. Each reservoir 234 is formed in accordance with any ofthe reservoir embodiments described herein.

As a result, each reservoir 234 can contain an accurately controlledamount of reagent which is to be transferred to a substrate. Further, acapillary can be embedded in the stamp member 190 to apply a biologicalreagent to the reservoir 234 of at least one of the plurality ofprojected portions 230. Also, a heating element and a temperature probecan be embedded in the stamp member 190 proximate to the reservoir 234to provide temperature control means for at least one of the projectedportions 230. The temperature control means can be utilized to provide ameans for amplifying a biological sample within the reservoir 234 of atleast one of the plurality of projected portions 230.

The projected portions in the above-described embodiments of stampmembers can be either fixed or selectively projected. Various means forselectively projecting at least one of the plurality of projectedportions can be utilized to allow a single stamp member to bereconfigured for a different pattern of locations on a substrate.

FIG. 13 illustrates an embodiment of a stamp member 240 havingselectively projectable transfer elements. A plurality of pistons 242 orother like translational mechanisms are included in the stamp member240. Each of the plurality of pistons 242 is independently projectableto form a respective projected portion. Preferably, each of theplurality of pistons 242 is electromechanically projectable. As aresult, a pattern of transfer elements for the stamp member 240 iselectronically programmable based upon a plurality of electricalsignals.

Selected ones of the plurality of pistons 242 are projected to form apattern of transfer elements. An absorbent layer 244 is deformed by theselected pistons which are projected. The absorbent layer 244 isutilized to receive a biological reagent for transfer to a substrate.This approach can be utilized for defining patterns having dimensions ofhundreds of microns, including dimensions as small as 100 μm and below.The use of selectively projectable transfer elements is advantageous inreducing a number of stamps required for a multilevel process.

FIG. 14 illustrates the stamp member 240 having an unprojected piston250 and a projected piston 252. The projected piston 252 deforms anabsorbent layer 254 to form a projected portion 256. The projectedportion 256 causes a biological reagent to be transferred to acorresponding location on a substrate 258 which contacts the stampmember 240.

FIG. 15 illustrates an embodiment of a selectively projectable stampmember having means for temperature control. Here, each of a pluralityof pistons 260 has a heating element 262 mounted thereto. Optionally, atemperature probe (not specifically illustrated) can also be mounted toeach of the plurality of pistons 260. The heating element 262 allows thetemperature of each of the projected portions to be controlled forpurposes described earlier.

In various embodiments of the present invention, the stamp member canhave various shapes including, but not limited to, a planar shape, acylindrical shape, a spherical shape, and other curved shapes. Thecylindrical shape is advantageous in that the stamp member can be rolledonto the substrate to transfer the at least one biological reagent fromthe plurality of transfer elements to the plurality of locations.

FIG. 16 shows an example of a stamp member 270 having a cylindricalshape. An outer circumferential surface 272 of the stamp member 270defines a plurality of transfer elements patterned to correspond to aplurality of locations on a substrate 274. Rolling the stamp member 270onto the substrate 274 causes a continuous pattern of a reagent to betransferred thereto.

FIG. 17 illustrates a roller 280 used to apply a reagent to a stampmember 282. The roller 280 is well-suited for applying a single type ofreagent to all of the plurality of transfer elements on the stamp member282. Use of the roller 280 is also advantageous to simultaneously applythe reagent to a portion of the stamp member 282 while another portionof the stamp member 282 is transferring the reagent to a substrate 284.Although the stamp member 282 is illustrated to have a cylindricalshape, it is noted that a roller can be utilized to apply a reagent tostamp members having a planar shape or another shape.

FIG. 18 illustrates at least one dispensing head 290 used to apply areagent to a stamp member 292. The at least one dispensing head 290applies a specific amount of the reagent directly to the plurality oftransfer elements on the stamp member 292. The at least one dispensinghead 290 is well-suited for applying different reagents to differenttransfer elements on the stamp member 292. As with the roller 280, theuse of the at least one dispensing head 290 is advantageous tosimultaneously apply the reagent to a portion of the stamp member 292while another portion of the stamp member 292 is transferring thereagent to a substrate 294.

In a preferred embodiment, the at least one dispensing head 290 isincluded within a plurality of dispensing bars as described in thecopending application entitled "Method and System for SynthesizingOligonucleotides using Nucleotide-Specific Dispensing Bars", now U.S.Pat. No. 5,733,509 which is incorporated by reference into thedisclosure of the present invention. Each of the plurality of dispensingbars is dedicated to dispensing a respective type of reagent. Forexample, each of four dispensing bars can be dedicated for applying arespective one type of four nucleotide types. Each of the plurality ofdispensing bars has a plurality of individually addressable dispensingheads to dispense a reagent in any of a row of transfer elements on thestamp member 292.

FIG. 19 is a flow chart summarizing steps performed in an embodiment ofmethod of placing at least one biological reagent at a plurality oflocations on a substrate. As indicated by block 300, the method includesa step of providing a stamp member which defines a plurality of transferelements patterned to correspond to the plurality of locations.Preferably, the stamp member is provided in accordance with thosedescribed herein, although alternative embodiments of the method are notlimited thereto.

If the plurality of transfer elements have the form of a plurality ofprojected portions of the stamp member, an optional step of selectivelyprojecting at least one of the plurality of projected portions can beperformed, as indicated by block 302. Each of the at least one of theplurality of projected portions can be electromechanically projected bya respective piston in the stamp member as described earlier. However,other means for selective projecting the at least one of the pluralityof projected portions can also be utilized.

As indicated by block 304, a step of applying the at least onebiological reagent to the stamp member is performed. For a transferelement which includes a reservoir, this step can include applying abiological reagent to the reservoir by a capillary embedded in the stampmember. For a plurality of reservoirs, this step can include applying arespective biological reagent to each of the plurality of reservoirs bya respective one of a plurality of capillaries embedded in the stampmember.

As indicated by block 306, a step of contacting the stamp member withthe substrate is performed to transfer the at least one biologicalreagent from the plurality of transfer elements to the plurality oflocations. As described earlier, the step of contacting the stamp memberwith the substrate can include either stamping or rolling the stampmember onto the substrate.

As indicated by block 308, an optional step of controlling a temperaturein a reservoir can be performed. This step can include applying a signalto a heating element in the stamp member proximate to the reservoir. Thetemperature can be controlled in this step to control a reaction whichoccurs in the reservoir.

As indicated by block 310, a step of maintaining contact between thestamp member and the substrate can be performed until a reactioninvolving the at least one biological reagent has completed. Thereafter,the stamp member is removed from the substrate.

As described earlier, this method is particularly advantageous where theat least one biological reagent includes at least one nucleotide, andwhere the plurality of locations are binding sites in a moleculardetection apparatus.

Thus, there has been described herein a concept, as well as severalembodiments including preferred embodiments of methods and systems forbiological reagent placement.

Because the various embodiments of the present invention utilize a stampmember which transfers a reagent by contacting a substrate, they providea significant improvement in that photolithographic patterning is notrequired to define chemically active areas on the substrate. This canincrease yield of a multilevel fabrication process by reducing a numberof steps involved. Further, the stamping process is scalable to largeareas and compatible with high-throughput manufacturing requirements.

The use of reservoirs as transfer elements reduces an amount of reagentneeded in comparison to immersing the entire substrate into a solution.The use of selectively projectable pistons to define the transferelements is advantageous in that the stamp member can be reconfigured inreal-time.

Additionally, the various embodiments of the present invention asherein-described allow chemical reactions, including hybridization, tobe accurately controlled by including providing temperature control ateach of the transfer elements. This reduces a requirement of uniformtemperature across an entire substrate.

Further, the various embodiments of the present invention allowdifferent samples to be placed only at predetermined regions on asubstrate, such as a molecular detection chip. Limiting sample placementcan reduce a likelihood of false-positive hybridization (or partialhybridization) in comparison to applying a sample to all of a pluralityof combinatorial sites on a molecular detection chip.

It will be apparent to those skilled in the art that the disclosedinvention may be modified in numerous ways and may assume manyembodiments other than the preferred form specifically set out anddescribed above.

Accordingly, it is intended by the appended claims to cover allmodifications of the invention which fall within the true spirit andscope of the invention.

What is claimed is:
 1. An apparatus for transferring at least onebiological reagent to a substrate, the apparatus comprising:a stampmember having a plurality of independently projectable portions; and anapplicator to apply the at least one biological reagent to at least oneof the independently projectable portions after the at least one of theindependently projectable portions has been selectively projected;wherein the at least one of the independently projectable portions isfor contacting with the substrate to transfer the at least onebiological reagent to the substrate.
 2. The apparatus of claim 1 whereinthe plurality of independently projectable portions includes a firstindependently projectable portion and a second independently projectableportion.
 3. The apparatus of claim 2 wherein the first independentlyprojectable portion is projected and the second independentlyprojectable portion is unprojected.
 4. The apparatus of claim 2 whereinthe stamp member includes a first electromechanical element associatedwith the first independently projectable portion and a secondelectromechanical element associated with the second independentlyprojectable portion.
 5. The apparatus of claim 4 wherein the firstelectromechanical element includes a first piston and wherein the secondelectromechanical element includes a second piston.
 6. The apparatus ofclaim 2 further comprising a first temperature probe associated with thefirst independently projectable portion and a second temperature probeassociated with the second independently projectable portion.
 7. Theapparatus of claim 2 further comprising a first heating elementassociated with the first independently projectable portion and a secondheating element associated with the second independently projectableportion.
 8. The apparatus of claim 1 wherein the applicator includes aroller.
 9. The apparatus of claim 1 wherein the applicator includes atleast one dispensing head.
 10. The apparatus of claim 1 wherein theapplicator comprises a capillary embedded in the stamp member.
 11. Amethod of transferring at least one biological reagent to a substrate,the method comprising the steps of:providing a stamp member having aplurality of independently projectable portions including a firstprojectable portion and a second projectable portion; selectivelyprojecting the first projectable portion; applying the at least onebiological reagent to the first projectable portion after selectivelyprojecting the first projectable portion; and contacting the stampmember with the substrate while the second projectable portion isunprojected to transfer the at least one biological reagent from thestamp member to the substrate.
 12. The method of claim 11 wherein thefirst projectable portion is projected in response to at least oneelectrical signal.
 13. The method of claim 11 wherein the stamp memberincludes a first electromechanical element associated with the firstindependently projectable portion and a second electromechanical elementassociated with the second independently projectable portion.
 14. Themethod of claim 13 wherein the first electromechanical element includesa first piston and wherein the second electromechanical element includesa second piston.
 15. The method of claim 11 further comprising:providinga first temperature probe associated with the first independentlyprojectable portion; and providing a second temperature probe associatedwith the second independently projectable portion.
 16. The method ofclaim 11 further comprising:providing a first heating element associatedwith the first independently projectable portion; and providing a secondheating element associated with the second independently projectableportion.